Combating the growth of neoplastic cells and tumors has been a major focus of biological and medical research. Such research has led to the discovery of novel cytotoxic agents potentially useful in the treatment of neoplastic disease. Examples of cytotoxic agents commonly employed in chemotherapy include anti-metabolic agents interfering with microtubule formation, alkylating agents, platinum-based agents, anthracyclines, antibiotic agents, topoisomerase inhibitors, and other agents.
Aside from merely identifying potential chemotherapeutic agents, cancer research has led to an increased understanding of the mechanisms by which these agents act upon neoplastic cells, as well as on other cells. For example, cholecalciferol (vitamin D) can effect differentiation and reduce proliferation of several cell types cells both in vitro and in vivo. The active metabolite of vitamin D (1,25-dihydroxycholecalciferol (hereinafter "1,25D.sub.3 ") and analogs (e.g., 1,25-dihydroxy-16-ene-23-yne-cholecalciferol (Ro23-7553), 1,25-dihydroxy-16-ene-23-yne-26,27-hexafluoro-19-nor-cholecalciferol (Ro25-6760), etc.) mediate significant in vitro and in vivo anti-tumor activity by retarding the growth of established tumors and preventing tumor induction (Colston et al., Lancet, 1, 188 (1989); Belleli et al., Carcinogenesis, 13, 2293 (1992); McElwain et al., Mol. Cell. Diff., 3, 31-50 (1995); Clark et al., J. Cancer Res. Clin. Oncol., 118, 190 (1992); Zhou et al., Blood, 74, 82-93 (1989)). In addition to retarding neoplastic growth, 1,25D.sub.3 induces a G.sub.0 /G.sub.1 -S phase block in the cell cycle (Godyn et al, Cell Proliferation, 27, 37-46 (1994); Rigby et al., J. Immunol., 135, 2279-86 (1985); Elstner et al., Cancer Res., 55, 2822-30 (1995); Wang et al., Cancer Res., 56, 264-67 (1996)). These properties have led to the successful use of 1,25D.sub.3 to treat neoplastic tumors (see Cunningham et al., Br. J. Cancer, 63, 4673 (1991); Mackie et al., Lancet, 342, 172 (1993), Bower et al., Proc. Am. Assoc. Cancer. Res., 32, 1257 (1991)).
In addition to its antineoplastic and cell-cycle blocking effects, 1,25D.sub.3 treatment can lead to hypercalcemia. As a result, 1,25D.sub.3 is typically administered for therapeutic applications (e.g., metabolic bone disease) at relatively low doses (e.g., about 1 .mu.g/day to about 2 .mu.g/day) long term. To mitigate the effects of hypercalcemia, analogs have been developed which retain antiproliferative activity without inducing hypercalcemia. (See, e.g., Zhou et al., Blood, 73, 75 (1991); Binderup et al., Biochem. Pharmacol., 42, 1569 (1991); Binderup et al., page 192 in Proceedings of the 8th Workshop on Vitamin D, Paris France (Norman, A. et al., Eds., Walter de Gruyter, Berlin, (1991))). Many of these synthetic analogs are more potent than 1,25D.sub.3 in inhibiting neoplastic growth (for a review of many such analogs, see Calverley et al., "Vitamin D" in Antitumor Steroids (Blickenstaff, R. T., Ed., Academic Press, Orlando (1992))).
The platinum-based agents are widely utilized in chemotherapeutic applications. For example, cisplatin kills tumor cells via formation of covalent, cross- or intrastrand DNA adducts (Sherman et al. Chem. Rev., 87, 1153-81 (1987); Chu, J. Biol. Chem., 269, 787-90 (1994)). Treatment with such platinum-based agents thereby leads to the inhibition of DNA synthesis (Howle et al., Biochem. Pharmacol., 19, 2757-62 (1970); Salles et al., Biochem. Biophys. Res. Commun., 112, 555-63 (1983)). Thus, cells actively synthesizing DNA are highly sensitive to cisplatin (Roberts et al., Prog. Nucl. Acid Res. Mol. Biol., 22, 71-133 (1979); Pinto et al., Proc. Nat. Acad. Sci. (Wash.) 82, 4616-19 (1985)). Such cells generally experience a growth arrest in G.sub.2 and eventually undergo apoptosis. This apoptotic effect is observed at drug concentrations insufficient to inhibit DNA synthesis (Sorenson et al., J. Natl. Cancer Inst., 82, 749-55 (1990)), suggesting that platinum agents act on neoplastic cells via multiple mechanisms. Some cells also demonstrate increased platinum sensitivity when in the G.sub.1 phase of the cell cycle (Krishnaswamy et al., Mutation Res., 293, 161-72 (1993); Donaldson et al., Int. J. Cancer, 57, 847-55 (1994)). Upon release from G.sub.0 /G.sub.1 -S block, such cells remain maximally sensitized through the remainder of the cell cycle.
Other chemotherapeutic agents act by different mechanisms. For example, agents interfering with microtubule formation (e.g., vincristine, vinblastine, paclitaxel, docetaxel, etc.) act against neoplastic cells by interfering with proper formation of the mitotic spindle apparatus (see, e.g., Manfredi et al., Pharmacol. Ther., 25, 83-125 (1984)). Thus, agents interfering with microtubule formation mainly act during the mitotic phase of the cell cycle (Schiff et al., Proc. Nat. Acad. Sci. U.S.A., 77, 1561-65 (1980); Fuchs et al., Cancer Treat. Rep., 62, 1219-22, (1978); Lopes et al., Cancer Chemother. Pharmacol., 32, 235-42 (1993)). Antimetabolites act on various enzymatic pathways in growing cells. For example, methotrexate (MTX) is a folic acid analog which inhibits dihydrofolate reductase. As a result, it blocks the synthesis of thymidylate and purines required for DNA synthesis. Thus, the primary impact of MTX is in the S phase of the cell cycle, but it can also impact RNA synthesis in G.sub.1 and G.sub.2 (Olsen, J. Am. Acad. Dermatol., 25, 306-18 (1991)).
Because of the differences in the biological mechanisms of various cytotoxic agents, protocols involving combinations of different cytotoxic agents have been attempted (e.g., Jekunen et al., Br. J. Cancer, 69, 299-306 (1994); Yeh et al., Life Sciences, 54, 431-35 (1994)). Combination treatment protocols aim to increase the efficacy of cytopathic protocols by using compatible cytotoxic agents. In turn, the possibility that sufficient antineoplastic activity can be achieved from a given combination of cytotoxic agents presents the possibility of reducing the dosage of individual cytotoxic agents to minimize harmful side effects. In part because the various cytotoxic agents act during different phases of the cell cycle, the success of combination protocols frequently depends upon the order of drug application (e.g., Jekunen et al., supra; Studzinski et al., Cancer Res., 51, 3451 (1991).
There have been attempts to develop combination drug protocols based, in part, on vitamin D derivatives. For example, the inhibitory effect of concurrent combination of 1,25D.sub.3 and platinum drugs on the growth of neoplastic cells has been studied (Saunders et al., Gynecol. Oncol., 51, 155-59 (1993); Cho et al., Cancer Res., 51, 2848-53 (1991)), and similar studies have focused on concurrent combinations of 1,25D.sub.3 and other cytotoxic agents (Tanaka et al., Clin. Orthopaed. Rel. Res. , 247, 290-96 (1989)). The results of these studies, however, have been less than satisfactory. In particular, the optimal sequence of drug administration has not been achieved. Moreover, the application of these approaches in therapy would require the long-term application of high doses of 1,25D.sub.3 in some protocols, which, as mentioned, can precipitate significant side effects. Thus, there remains a need for an improved method of enhancing the efficacy of chemotherapeutic agents, particularly a need for an improved combination therapy, especially involving vitamin D derivatives.